Fluorescence occurs in certain molecules called fluorophores or fluorescent dyes in three sequential stages involving excitation, excited state lifetime, and fluorescence emission. When an excited fluorophore is raised to a singlet state, it decays back to ground state by emission of a photon that generates a fluorescent signal. Generally, fluorescent dyes absorb light at a particular wavelength and emit light at a wavelength longer than that absorbed. The difference between the absorption and emission wavelength maxima is known as the Stokes shift (Handbook of Fluorescent Probes and Research Products, Molecular Probes, Eugene, Oreg., Haughland, 2002). Large Stokes shifts and fluorescence emission at long wavelengths are viewed as practically useful to overcome the problem of fluorescence measurement in the presence of background signals such as, Raman scattering and auto fluorescence (e.g. of plastic and biological materials). Only a very few individual fluorescent dyes have large Stokes shifts. However, large Stokes shifts can result from fluorescent dye pairs. In the presence of other molecules, including dyes, the fluorescence of most fluorophores is typically quenched, whereas in some cases, fluorescence at longer wavelengths may result. Three different types of such fluorescence from dye pairs have been described.
Excimers and exciplexes are electronically excited dimer complexes which are non-binding in the ground state. Excimers and exciplexes complexes can be formed between dye molecules, whose close proximity is typically less than a few nm. The formation of such complexes effectively results in fluorescence with a large Stokes shift J. Phys. Chem., 100, (1996)11539-11545. In the case of excimers, a pair of dye molecules form a complex by the interaction of an excited molecular entity with a ground state partner of the same structure. The close proximity between such molecules results in energy transfer and fluorescence with a large Stokes shift. In the case of exciplexes, a pair of dye molecules (sometimes only one being a fluorescent dye) form a complex between an excited molecular entity with a ground state partner of a different chemical structure. The molecules are in very close proximity to transfer energy. The close proximity affects fluorescence properties. For instance at high concentrations, or when linked on a short spacer, two pyrene molecules are in the vicinity of each other for the π-systems to overlap causing a fluorescence emission maximum at a longer wavelength (about 470 nm) than at low concentrations where the pyrene molecules are too far apart as monomers and only an emission at 378 and 396 nm is observed.
Fluorescence resonance energy transfer (FRET) is a technique in which the energy emitted from one fluorophore (the donor) causes the excitation of a second, longer wavelength, fluorophore (the acceptor). The transfer of the excitation energy of the donor to the acceptor molecule is only possible if the electronic transition of the donor from the excited state to the ground state corresponds to the absorption wavelength of the acceptor. This requires substantial overlap of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor molecule. The combination of fluorophores to form FRET pairs is limited by the requirement for the donor's emission spectra to overlap with acceptor's excitation spectra. In addition to this, the transition dipoles of donor and acceptor need to be correctly orientated. (Matyus, 1992, J. Photochem. Photobiol. B: Biol., 12: 323-337). In contrast to excimers and exciplex fluorescence, FRET pairs do not require the dye molecules forming the complexes to be in very close proximity as FRET can arise at up to 10 nm distance although typically the range is 4-6 nm for favourable pairs of donor and acceptor dyes. FRET is commonly used in several detection modes to measure or identify a variety of biologically active molecules including nucleic acids, oligonucleotides, and proteins. One of the advantages of FRET is that fluorescence arises under physiological conditions in comparison to exciplex fluorescence which is typically weak under aqueous conditions, requiring the addition of organic solvents or formation in a similar molecular microenvironment.
A fluorescent chemosensor modulates its emission properties upon binding of an analyte to a receptor. Fluorescence properties have been used to provide information on ligand binding, ligand or probe environment, and conformational changes. A number of homogeneous assay systems, which use fluorescence as detection means are based on polarization, lifetimes, quenching, and energy transfer schemes (Drug Discovery Today (2003) Vol. 8, No. 22 1035-1043).
Peptides doubly tagged with fluorescent dyes (Biophys. Chem. 67(1997), 167-176) have previously been used as fluorogenic substrates for proteinases. In these assays dye-to-dye contact diminishes the fluorescence of the participating dyes by quenching. On enzymatic cleavage of the peptide link, the dye-tagged products dissociate, breaking dye to dye contact, thus relieving quenching of the fluorescence. To observe the increase in fluorescence indicative of enzyme activity usually requires breaking of a covalent bond in the linker. Fluorescent quenching has been used (Analytical Biochemistry 165(1987) 96-101) to measure the distance between a quencher and a fluorophore when attached to a peptide linker. Ai-Ping Wei et al (WO95/03429) uses antibody-antigen reaction to break dye-to-dye contact in order that molecules in the dimer state (fluorescence quenched) become monomeric (fluorescence unquenched) to relieve quenching. This was used to form assays measuring specific antibodies to a recognized peptidic epitope that linked the two dyes. In common with many other homogeneous dequenching assays, while this method can measure antibodies specific to the epitope (used to bind the dyes) in a noncompetitive manner, its adaptation to measuring other analytes, possible only in competitive mode, suffers from disadvantage in that the fluorescence signal becomes indirectly proportional to analyte concentration.
Pyrenyl derivatized peptides have been successfully used to investigate peptidic structures (Org. Lett, Vol. 3, No. 16, 2001). When pyrenes are separated as monomers, chromophores display an emission band with distinct vibrational structure between 370 and 430 nm; a broad vibrationless (excimer) band centered around 470 nm is observed when pyrenes are in close proximity. In addition, the ground-state aggregation of the chromophores leads to perturbation in the UV/vis absorption. An assay using fluorogenic peptides based on the monomer/excimer (Analytical Biochemistry 306(2002), 247-251) fluorescence features of pyrene was developed to measure the proteolytic activity of trypsin. Two pyrene moieties incorporated into the respective N- and C-terminus of the peptides led to an expected increase in monomer fluorescence and a decrease in excimer fluorescence of pyrene as the peptide is hydrolysed by the enzyme. In another assay (Bioconjug Chem. (1997) 8, 560-6) streptavidin binding to a biotin labeled pyrene derivative causes the appearance of the excimer emitting at 470 nm. The ratio of monomer to excimer then provides the concentration of unlabeled biotin in the sample. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. In yet another assay system (U.S. Pat. No. 5,314,802) the excimer can be formed by assembling two pyrenes in close proximity using an antibody and this was used in a competitive manner with analyte modified pyrene analogues to measure free analyte. Pyrene excimer has also been used in FRET assays where the energy transfer from the excimer emission (470 nm) to BODIPY-FL-GM1 was anticipated by the good overlap between pyrene excimer fluorescence and absorption spectrum of BODIPY-FL-GM1 in lipid vesicles (Langmuir 1999, 15, 4710-4712). By using pyrene-containing lipids, the intensity of the excimer peak has been used to report lipid redistribution in liposomes (Chem. Phys. Lipids 2000, 106, 89-99). Pyrene is a hydrophobic molecule whose fluorescence efficiency is susceptible to solvent polarity. The fluorescence lifetime of pyrene is significantly longer and this property has been used in number of studies (Journal of Biochemistry (1982) Vol 92, 1425-1430) to probe microenvironment. Both pyrene monomer and excimer fluorescence has been used (Nucleic Acids Res. 26(1998), 5409-5416, U.S. Pat. No. 5,332,659) as an indicator for monitoring DNA hybridisation. Hybridisation of two oligonucleotides labelled by a single pyrene group at the terminal ends with complementary DNA results in the excimer formation.
Molecular Beacons (Nature Biotechnology 14 (1996), pp. 303-308) contain a fluorophore and a quencher linked in a stem-loop structure. The stem sequence maintains dyes in close proximity so that photons emitted by the fluorophore are quenched and not emitted. The loop sequence hybridises with the target giving the spatial separation of the fluorophore from the quencher, allowing the fluorescence to appear and be measured. HyBeacons (International Patent Application No. PCT/GB01/01430) uses a single probe, in the absence of a quencher moiety, enhancing fluorescence when bound to complementary target DNA sequences than when the probes are in the single-stranded conformation. This shift in the quantity of fluorescence emission occurs as a direct result of target hybridization permitting the detection of DNA sequences.